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. 1993 May;67(5):2628–2636. doi: 10.1128/jvi.67.5.2628-2636.1993

Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

D P Grandgenett 1, R B Inman 1, A C Vora 1, M L Fitzgerald 1
PMCID: PMC237584  PMID: 8474165

Abstract

Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowerman B., Brown P. O., Bishop J. M., Varmus H. E. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. doi: 10.1101/gad.3.4.469. [DOI] [PubMed] [Google Scholar]
  2. Brown P. O., Bowerman B., Varmus H. E., Bishop J. M. Correct integration of retroviral DNA in vitro. Cell. 1987 May 8;49(3):347–356. doi: 10.1016/0092-8674(87)90287-x. [DOI] [PubMed] [Google Scholar]
  3. Bushman F. D., Fujiwara T., Craigie R. Retroviral DNA integration directed by HIV integration protein in vitro. Science. 1990 Sep 28;249(4976):1555–1558. doi: 10.1126/science.2171144. [DOI] [PubMed] [Google Scholar]
  4. Craigie R., Fujiwara T., Bushman F. The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro. Cell. 1990 Aug 24;62(4):829–837. doi: 10.1016/0092-8674(90)90126-y. [DOI] [PubMed] [Google Scholar]
  5. Engelman A., Mizuuchi K., Craigie R. HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer. Cell. 1991 Dec 20;67(6):1211–1221. doi: 10.1016/0092-8674(91)90297-c. [DOI] [PubMed] [Google Scholar]
  6. Farnet C. M., Haseltine W. A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol. 1991 Apr;65(4):1910–1915. doi: 10.1128/jvi.65.4.1910-1915.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farnet C. M., Haseltine W. A. Integration of human immunodeficiency virus type 1 DNA in vitro. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4164–4168. doi: 10.1073/pnas.87.11.4164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fitzgerald M. L., Vora A. C., Grandgenett D. P. Development of an acid-soluble assay for measuring retrovirus integrase 3'-OH terminal nuclease activity. Anal Biochem. 1991 Jul;196(1):19–23. doi: 10.1016/0003-2697(91)90111-6. [DOI] [PubMed] [Google Scholar]
  9. Fitzgerald M. L., Vora A. C., Zeh W. G., Grandgenett D. P. Concerted integration of viral DNA termini by purified avian myeloblastosis virus integrase. J Virol. 1992 Nov;66(11):6257–6263. doi: 10.1128/jvi.66.11.6257-6263.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fujiwara T., Craigie R. Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration. Proc Natl Acad Sci U S A. 1989 May;86(9):3065–3069. doi: 10.1073/pnas.86.9.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fujiwara T., Mizuuchi K. Retroviral DNA integration: structure of an integration intermediate. Cell. 1988 Aug 12;54(4):497–504. doi: 10.1016/0092-8674(88)90071-2. [DOI] [PubMed] [Google Scholar]
  12. Funnell B. E., Inman R. B. Comparison of partial denaturation maps with the known sequence of simian virus 40 and phi X174 replicative form DNA. J Mol Biol. 1979 Jun 25;131(2):331–340. doi: 10.1016/0022-2836(79)90079-2. [DOI] [PubMed] [Google Scholar]
  13. Grandgenett D. P., Vora A. C., Schiff R. D. A 32,000-dalton nucleic acid-binding protein from avian retravirus cores possesses DNA endonuclease activity. Virology. 1978 Aug;89(1):119–132. doi: 10.1016/0042-6822(78)90046-6. [DOI] [PubMed] [Google Scholar]
  14. Grandgenett D. P., Vora A. C. Site-specific nicking at the avian retrovirus LTR circle junction by the viral pp32 DNA endonuclease. Nucleic Acids Res. 1985 Sep 11;13(17):6205–6221. doi: 10.1093/nar/13.17.6205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Inman R. B., Schnös M. Partial denaturation of thymine- and 5-bromouracil-containing lambda DNA in alkali. J Mol Biol. 1970 Apr 14;49(1):93–98. doi: 10.1016/0022-2836(70)90378-5. [DOI] [PubMed] [Google Scholar]
  16. Jones K. S., Coleman J., Merkel G. W., Laue T. M., Skalka A. M. Retroviral integrase functions as a multimer and can turn over catalytically. J Biol Chem. 1992 Aug 15;267(23):16037–16040. [PubMed] [Google Scholar]
  17. Katz R. A., Merkel G., Kulkosky J., Leis J., Skalka A. M. The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro. Cell. 1990 Oct 5;63(1):87–95. doi: 10.1016/0092-8674(90)90290-u. [DOI] [PubMed] [Google Scholar]
  18. Khan E., Mack J. P., Katz R. A., Kulkosky J., Skalka A. M. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. doi: 10.1093/nar/19.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kitamura Y., Lee Y. M., Coffin J. M. Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5532–5536. doi: 10.1073/pnas.89.12.5532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Knaus R. J., Hippenmeyer P. J., Misra T. K., Grandgenett D. P., Müller U. R., Fitch W. M. Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA. Biochemistry. 1984 Jan 17;23(2):350–359. doi: 10.1021/bi00297a026. [DOI] [PubMed] [Google Scholar]
  21. Leavitt A. D., Rose R. B., Varmus H. E. Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae. J Virol. 1992 Apr;66(4):2359–2368. doi: 10.1128/jvi.66.4.2359-2368.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee Y. M., Coffin J. M. Efficient autointegration of avian retrovirus DNA in vitro. J Virol. 1990 Dec;64(12):5958–5965. doi: 10.1128/jvi.64.12.5958-5965.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Littlewood R. K., Inman R. B. Computer-assisted DNA length measurements from electron micrographs with special reference to partial denaturation mapping. Nucleic Acids Res. 1982 Mar 11;10(5):1691–1706. doi: 10.1093/nar/10.5.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Misra T. K., Grandgenett D. P., Parsons J. T. Avian retrovirus pp32 DNA-binding protein. I. Recognition of specific sequences on retrovirus DNA terminal repeats. J Virol. 1982 Oct;44(1):330–343. doi: 10.1128/jvi.44.1.330-343.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mumm S. R., Horton R., Grandgenett D. P. v-Src enhances phosphorylation at Ser-282 of the Rous sarcoma virus integrase. J Virol. 1992 Apr;66(4):1995–1999. doi: 10.1128/jvi.66.4.1995-1999.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pryciak P. M., Sil A., Varmus H. E. Retroviral integration into minichromosomes in vitro. EMBO J. 1992 Jan;11(1):291–303. doi: 10.1002/j.1460-2075.1992.tb05052.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roth M. J., Schwartzberg P. L., Goff S. P. Structure of the termini of DNA intermediates in the integration of retroviral DNA: dependence on IN function and terminal DNA sequence. Cell. 1989 Jul 14;58(1):47–54. doi: 10.1016/0092-8674(89)90401-7. [DOI] [PubMed] [Google Scholar]
  28. Sandmeyer S. B., Bilanchone V. W., Clark D. J., Morcos P., Carle G. F., Brodeur G. M. Sigma elements are position-specific for many different yeast tRNA genes. Nucleic Acids Res. 1988 Feb 25;16(4):1499–1515. doi: 10.1093/nar/16.4.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sherman P. A., Fyfe J. A. Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5119–5123. doi: 10.1073/pnas.87.13.5119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shih C. C., Stoye J. P., Coffin J. M. Highly preferred targets for retrovirus integration. Cell. 1988 May 20;53(4):531–537. doi: 10.1016/0092-8674(88)90569-7. [DOI] [PubMed] [Google Scholar]
  31. Terry R., Soltis D. A., Katzman M., Cobrinik D., Leis J., Skalka A. M. Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. J Virol. 1988 Jul;62(7):2358–2365. doi: 10.1128/jvi.62.7.2358-2365.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vink C., Yeheskiely E., van der Marel G. A., van Boom J. H., Plasterk R. H. Site-specific hydrolysis and alcoholysis of human immunodeficiency virus DNA termini mediated by the viral integrase protein. Nucleic Acids Res. 1991 Dec 25;19(24):6691–6698. doi: 10.1093/nar/19.24.6691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vink C., van Gent D. C., Elgersma Y., Plasterk R. H. Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage. J Virol. 1991 Sep;65(9):4636–4644. doi: 10.1128/jvi.65.9.4636-4644.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vink C., van Gent D. C., Plasterk R. H. Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells. J Virol. 1990 Oct;64(10):5219–5222. doi: 10.1128/jvi.64.10.5219-5222.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vora A. C., Fitzgerald M. L., Grandgenett D. P. Removal of 3'-OH-terminal nucleotides from blunt-ended long terminal repeat termini by the avian retrovirus integration protein. J Virol. 1990 Nov;64(11):5656–5659. doi: 10.1128/jvi.64.11.5656-5659.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Woerner A. M., Klutch M., Levin J. G., Marcus-Sekura C. J. Localization of DNA binding activity of HIV-1 integrase to the C-terminal half of the protein. AIDS Res Hum Retroviruses. 1992 Feb;8(2):297–304. doi: 10.1089/aid.1992.8.297. [DOI] [PubMed] [Google Scholar]

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